Surface-expressed invariant chain (CD74) is required for internalization of human leucocyte antigen-DR molecules to early endosomal compartments

Abstract

Transport of major histocompatibility complex (MHC) class II molecules to the endocytic route is directed by the associated invariant chain (Ii). In the endocytic pathway, Ii is proteolytically cleaved and, upon removal of residual Ii fragments, class II alpha beta dimers are charged with antigenic peptide and recognized by CD4+ T cells. Although distinct peptide-loading compartments such as MIIC (MHC class II loading compartment) and CIIV (MHC class II vesicles) have been characterized in different cells, there is growing evidence of a multitude of subcellular compartments in which antigenic peptide loading takes place. We employed a physiological cellular system in which surface Ii (CD74) and surface human leucocyte antigen (HLA)-DR were induced either alone or in combination. This was achieved by transient exposure of HT-29 cells to recombinant interferon-gamma (rIFN-gamma). Using distinct cellular variants, we showed that: (i) the majority of Ii molecules physically associate on the cell membrane with class II dimers to form DR alpha beta:Ii complexes; (ii) the presence of surface Ii is a prerequisite for the rapid uptake of HLA-DR-specific monoclonal antibodies into early endosomes because only the surface DR+/Ii+ phenotype, and not the DR+/Ii- variant, efficiently internalizes; and (iii) the HLA-DR:Ii complexes are targeted to early endosomes, as indicated by co-localization with the GTPase, Rab5, and endocytosed bovine serum albumin. Internalization of HLA-DR:Ii complexes, accommodation of peptides by DR alphabeta heterodimers in early endosomes and recycling to the cell surface may be a mechanism used to increase the peptide repertoire that antigen-presenting cells display to MHC class II-restricted T cells.

Figure 1

Surface expression of major histocompatibility complex (MHC) class I molecules, Ii and human leucocyte antigen (HLA)-DR on interferon-γ (IFN-γ)-treated HT-29 cells. HT-29 cells cultured in the presence of the indicated concentrations of rIFN-γ (0–500 U/ml) for 72 hr were analysed by flow cytometry using the following mAbs: W6/32 (anti-HLA-ABC framework), BU45 (reacting with a C-terminal/extracellular determinant of all Ii isoforms) and L243 (reacting with mature HLA-DR molecules), represented by filled histograms, respectively. As a negative control, HT-29 cells were stained with the mAb VIC-Y1 (reacting with a cytoplasmic determinant of Ii, outlined histograms).

Figure 2

Kinetics of the interferon-γ (IFN-γ)-driven induction of Ii and human leucucyte antigen (HLA)-DR surface expression on HT-29 cells. HT-29 cells exposed for different lengths of time to 100 U/ml rIFN-γ were analysed by flow cytometry using saturating amounts of mAbs: BU45 (filled circles) and L243 (open circles). The results are depicted as percentage positive cells above the cut-off value of background fluorescence determined with the control mAb VIC-Y1. Results are the average of three independent experiments, the error bars representing the standard deviation.

Figure 3

Four distinct phenotypes of HT-29 cells are generated by sequential induction and decline of DR and Ii. Surface expression of human leucocyte antigen (HLA)-DR and Ii molecules was measured by flow cytometry after the indicated time of culture in the presence of, or after removal of, recombinant interferon-γ (rIFN-γ) using mAbs W6/32, BU45, and L243(filled histograms). mAb VIC-Y1 (recognizing a cytoplasmic epitope of Ii) was used as a negative control (outlined histograms).

Figure 4

Two-dimensional separation of human leucocyte antigen (HLA)-DR and Ii immunoprecipitates. HT-29 cells induced with 100 U/ml recombinant interferon-γ (rIFN-γ) for 72 hr were surface labelled with ¹²⁵I. Invariant chains were immunoprecipitated with BU45 (a) and HLA-DR molecules with TAL.1B5 (b). Samples were separated in the first dimension by isoelectric focusing (acidic end to the right side) and in the second dimension by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). Although DRα and DRβ spots are very prominent, no Ii spots were detectable, reflecting the fact that the surface expression of HLA-DR molecules is much higher than that of Ii.

Figure 5

Localization of mAb after exposure to living cells. HT-29 cells, either stimulated for 24 hr with 100 U/ml recombinant interferon-γ (rIFN-γ) (Ii⁺DR⁺ phenotype) or, alternatively, pretreated for 120 hr with 100 U/ml rIFN-γ followed by 4 days of culture without rIFN-γ (Ii⁻DR⁺ phenotype), were detached and incubated for 20 min at 37° in the presence of saturating amounts of the following mAbs: BU45, DA6·231 and L243. Subsequently, cells were placed on ice, non-bound mAb was removed by washing in cold medium, and internalized antibody was detected by immunocytochemical staining of acetone-fixed cytospin preparations. The difference in average cell size between Ii⁺/DR⁺ and Ii⁻/DR⁺ cells is a result of the effect of rIFN-γ.

Figure 6

(a) Rapid turnover of Ii on the surface of recombinant interferon-γ (rIFN-γ)-treated HT-29 cells. For internalization of surface-bound BU45, HT-29 cells pretreated for 72 hr with rIFN-γ were labelled with saturating amounts of BU45 on ice for 1 hr. After removing non-bound mAb, 50 μl of suspension containing 10⁶ cells was added to 450 μl of medium, prewarmed to 37°, to allow endocytosis for different periods of time. Subsequently, cells were recooled by transfer to 4·5 ml of ice-cold medium and analysed for remaining surface-bound mAb by flow cytometry. Internalization of BU45 is depicted as the percentage of surface-bound mAb remaining after endocytosis at 37° relative to the total amount of mAb bound to cells that were kept on ice throughout. As a control, internalization of prebound BU45 was analysed under hyperosmotic conditions (0·45 m sucrose) known to inhibit coated pit-mediated endocytosis. The depletion of Ii from the plasma membrane at at steady state was measured after blocking transport of newly synthesized Ii by treatment with 1 μg/ml brefeldin A (BFA). Cells were preincubated with BFA for 15 min on ice followed by shifting the cells to 37° in the presence of BFA for different periods of time. The results are expressed as the percentage of Ii surface levels remaining after BFA treatment at 37° relative to the Ii level on cells not shifted to 37°. (b) HT-29 cells grown on slides were exposed to 100 U/ml IFN-γ for 24 hr. Control cells (Control) were incubated with saturating amounts of BU45 (50 μg/ml) in normal medium for 20 min at 37°. Alternatively, cells were preincubated for 10 min at 37° in hypertonic medium containing 0·45 m sucrose followed by further incubation for 20 min at 37° in the presence of 50 μg/ml BU45 diluted in the same sucrose-containing medium (0·45 m sucrose). Non-bound mAb was removed by rinsing in cold medium; subsequently, cells were acetone fixed and surface bound, and cytoplasmically located antibody was detected by immunocytochemical staining.

Figure 7

Multicolour immunofluorescence microscopic analysis of antibody internalization. HT-29 cells grown on glass slides were stimulated for 36 hr with 500 U/ml recombinant interferon-γ (rIFN-γ). The antibodies were added simultaneously in various combinations. Absorption of antibodies was performed on ice for 30 min, after which the cells were washed and warmed at 37° to allow internalization for 10 min prior to acteone fixation. Bound and processed antibodies were then detected via isotype-specific goat antimouse antibodies carrying different fluorochromes. By combining filters, each spectral component of the image could be visualized individually or superimposed (rows 1–3). In an extension of these experiments, cells were additionally exposed to biotin-labelled bovine serum albumin (BSA; rows 4 and 5). Internalized BSA was detected via AMCA-conjugated streptavidin. Arrows mark identical areas of interest in different sets of filter combinations. First row: cells co-incubated with BU43 and L227 at 4° and then warmed to 37° show internalization of BU43 (a, red), L227 (b, green) in a co-localized manner (c: BU43/L227, yellow). Second row: cells co-incubated with BU43 and L243, and then treated as described above, show selective uptake of BU43 (d), while bound L243 antibodies remain at the cell surface (e) and therefore do not co-localize (f: BU43/L243). Third row: experimental setting as described for the first row except under hyperosmotic conditions. Antibody internalization of BU43 (g), L227 (h) is completely blocked resulting in their co-localization at the cell surface (i: BU43/L227). Fourth row: cells exposed to BSA at 37° for 10 min were acetone fixed to allow anti-Rab-5 antibody to penetrate the cells; Rab5 (k) and internalized BSA (l) are co-localized in early endosomes (m). Fifth row: in the presence of BSA, cells were exposed simultaneously to BU43 and L227. Co-internalized antibodies BU43 (n) and L227 (o) are targeted to the subcellular sites where BSA accumulates (p) resulting in co-localizion of BU43/L227/BSA (q). For enhancement of contrast, the AMCA blue in Fig. 1 and (p) has been changed to the pseudocolor white.